Catalase-inactivating compounds and the use thereof

ABSTRACT

The invention relates to the sulfonyl esters of the general formula R 1 —COO—SO 2 —Z—R 2 , wherein Z, R 1  and R 2  are defined as in claim  1 . The invention further relates to the use of one of the inventive compounds for modifying the kinetics of the enzymatic effect of catalase. A method for measuring a concentration in living and/or active microorganisms in a liquid sample ( 3 ) by means of the development of oxygen from hydrogen peroxide is also based on the above-mentioned modification. An inventive compound is admixed in a container ( 1 ) to the sample, said compound inactivates the enzymatic effect of endogenous catalase without substanially inactivating the enzymatic effect of the intracellular catalase of microorganisms. Hydrogen peroxide is added and immediately afterwards the pressure present in the sample container is briefly equalized with the atmosphere, the container is closed in a gas-tight manner ( 2 ) during a predetermined reaction time and the pressure in the container is measured ( 9, 10, 11 ). The pressure measured and the data regarding the microorganisms and the sample dilution are used to calculate ( 22 ) the concentration of living and/or active microorganism in the sample and the concentration is optionally displayed ( 24 ). The invention further relates to a device for carrying out the inventive method.

RELATED APPLICATION

This Application is a 371 of PCT/CH02/00270 May 17, 2002 published as WO03/004462A1 on Jan. 16, 2003.

TECHNICAL FIELD

The invention relates to sulfonyl esters of the general formulaR₁—COO—SO₂—Z—R₂ in which Z, R₁ and R₂ have the meanings defined in claim1, to a use of these compounds for modifying the kinetics of theenzymatic action of catalase, to a method for measuring a concentrationof live and/or active microorganisms such as bacteria and/or fungi,especially molds and/or yeasts, in a liquid sample by means of theevolution of oxygen from hydrogen peroxide, and to an apparatus forcarrying out this method.

PRIOR ART

Determination of the microbial contamination of foodstuffs, coolinglubricants and the like, or else the investigation of the efficacy of abiocide or disinfectant, currently takes place mainly by microbiologicalmethods which make use of a growth of the microorganisms to bedetermined and are therefore slow and unsuitable for continuousmonitoring of the microbial contamination on site.

The principal causes of microbial contamination are in the case offoodstuffs mainly aerobic microorganisms (such as bacteria and/or fungi,especially molds and/or yeasts) and in the case of aqueous coolinglubricant emulsions also microorganisms which are facultative anaerobes.All microorganisms which have a respiratory system based on cytochromeelectron transport to generate energy (and these are almost allmicroorganisms occurring in foodstuffs and cooling lubricant emulsions)have a particular enzyme called catalase which chemically is atetrameric iron-protein (M_(r)=245000) with 4 heme mol in the form offerriprotoporphyrin IX with iron(III). Catalase extremely rapidlycleaves hydrogen peroxide into water and oxygen, and the oxygenevolution is directly proportional to the enzyme concentration. This isthe basis for a known method for determining the microbialcontamination, which is faster than methods dependent on growth ofmicroorganisms—compare in this connection H. K. Frank & U.Hertkorn-Obst, Chem. Mikrobiol. Technol. Lebensm. 6, 143–149 (1980)and/or R. G. Kroll, E. R. Frears & A. Bayliss, J. Appl. Bacteriol. 66,209–217 (1989). Measurement of the oxygen evolution is possible invarious known ways, inter alia by manometry—compare in this connectionG. J. Wang and D. Y. C. Fung, J. of Food Safety 8, 46–47 (1986) andEP-476850. It is known to be advantageous to put the microorganisms tobe determined into an aqueous sample and to add hydrogen peroxide in anamount resulting in a concentration of about 1% by volume, the optimalpH for the measurement for most microorganisms to be determined being inthe neutral region at 7.

Another requirement for determining the microbial contamination of aninvestigated material or a sample thereof is to detect only the livemicroorganisms, i.e. only the enzymatic action of the activeintracellular catalase of these live microorganisms is important andrequires measurement. However, measurement errors result from theenzymatic action of active endogenous catalase from a wide variety ofsources present in the sample. Thus, various difficulties are reportedboth by H. K. Frank & U. Hertkorn-Obst and by R. G. Kroll, E. R. Frears& A. Bayliss. A special effort must be made to distinguish between liveand dead yeast cells (brewer's yeast). Foodstuffs contain non-microbialendogenous catalase which is introduced into the sample by plantconstituents (lettuce, onions), juice (apple juice, lemon juice) andanimal tissue (erythrocytes in meat and milk), and markedly falsifiesthe measurement through its enzymatic action unless the action of thisnon-microbial endogenous catalase has been previously eliminated forexample by heat treatment (pasteurization, preservation). Drinking waterand superfluous water also contain interfering plant residues andsuspended matter of a wide variety of types, which catalyze oxygenevolution as soon as hydrogen peroxide is introduced into the sample,unless these interfering substances are eliminated by filtration.

The abovementioned difficulties make the currently available, relativelyfast methods for determining the microbial contamination of foodstuffs,cooling lubricants and the like, as well as for investigating theactivity of a biocide or disinfectant, unreliable.

EP-184260 discloses a method with which the total catalase content, butnot the concentration of live and/or active microorganisms is detected.In this method, both the bacterial and the non-bacterial, i.e.endogenous, catalase is detected, leading to considerable differences.Thus, owing to the imperfect relationship between the number ofcolony-forming units (CFU) and the catalase levels under the influenceof biomass, an assessment of the microbial contamination or measurementof the concentration of live and/or active microorganisms is notpossible in every case.

U.S. Pat. No. 5,610,025 and/or WO-93/15218 discloses a method with whichhydrogen peroxide-decomposing enzymes are detected in the presence ofcatalase. Addition of the hydrogen peroxide is preceded by inhibition ofcatalase by addition of a hydroxylamine salt. This teaching does notlead to a measurement of the concentration of live and/or activemicroorganisms.

It is accordingly an object of the invention to provide a method formeasuring a concentration of live and/or active microorganisms whosemeasured results are reliable and, in particular, are not falsified byendogenous catalase.

It is accordingly also an object of the invention to indicate andprovide chemical compounds which bring about a modification of thekinetics of the enzymatic action of catalase, in particular a preferablyirreversible inactivation of the enzymatic action of catalase, inparticular of active endogenous catalase, in particular withoutnoticeable inactivation of the enzymatic action of active intracellularcatalase.

It is likewise an object of the invention to provide a method of theaforementioned type, whose measured results can be obtained andinterpreted rapidly, and an apparatus suitable therefor.

DESCRIPTION OF THE INVENTION

To achieve these and further objects which are evident from thefollowing description of the invention, novel chemical compounds of theinvention are indicated and their uses according to the invention aredefined in the corresponding product claims and use claims.

A method according to the invention and preferred further developmentsthereof are likewise defined in the corresponding method claims, and apreferred apparatus according to the invention for carrying out themethod according to the invention and preferred further developmentsthereof are defined in the corresponding apparatus claims.

Owing to the simplicity of the method according to the invention,especially in connection with the apparatus according to the inventionfor carrying it out, the invention makes it possible to carry out themethod easily, rapidly and cost-effectively, with direct indication of aconcentration of live and/or active microorganisms being made possible.

Since both the reaction time and the measurement time are short in themethod according to the invention, there is the possibility for exampleof subjecting a foodstuff immediately before marketing or shortly beforeconsumption to a check so that batches with a microbial contaminationwhich is too high can be withheld in good time.

In particular, the measured results of the method according to theinvention can be obtained within a shorter time than, for example, halfan hour and therefore currently (virtually in real time), making usethereof possible on site for controlling and/or monitoring a method,where the active intracellular catalase is that of live microorganismssuch as bacteria and/or fungi, especially molds and/or yeasts.

In addition, the chemical compounds indicated and provided by theinvention bring about a modification of the kinetics of the enzymaticaction of catalase, which makes it possible to use them for killingmicroorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments of the invention are described in detail below bymeans of the drawings. These show:

FIG. 1 to 8 consecutive steps in a method according to the invention bymeans of diagrammatic depictions of articles and liquids used therefor;

FIG. 9 a diagrammatically depicted pressure gage of an apparatus forcarrying out the method according to the invention having a pressureprobe part and a pressure evaluation part each depicteddiagrammatically;

FIG. 10 a diagrammatically depicted ventilation implement of anapparatus for carrying out the method according to the invention; and

FIG. 11 a diagrammatically depicted stopper for use with an apparatusfor carrying out the method according to the invention.

WAYS OF CARRYING OUT THE INVENTION

It has been found within the scope of the invention that particularchemical compounds are able to modify the kinetics of the enzymaticaction of catalase. This modification may be different with activeendogenous catalase or active intracellular catalase. Depending on thechemical compound used and on the type of catalase modified thereby(endogenous or intracellular, in the latter case present in cells ofdifferent microorganisms), this modification may effect irreversibleinactivation of all the catalase, and/or an irreversible inactivation ofactive endogenous catalase without noticeable inactivation of theenzymatic action of active intracellular catalase can be achieved. Avariety of applications derive therefrom, among which the measurement ofa concentration of live and/or active microorganisms and/or the killingof microorganisms are of particular interest.

Microorganisms suitable in this connection may be bacteria and/or fungi,especially molds and/or yeasts.

Chemical compounds suitable in this connection are sulfonyl esters ofthe general formulaR₁—COO—SO₂—Z—R₂in which

-   -   Z is phenylene or is absent,    -   R₁ is connected via a primary or secondary carbon atom to the        carbon atom of the acid radical —COO— and is straight-chain or        branched alkyl having 2 to 21000 carbon atoms, straight-chain or        branched alkenyl having 2 to 21000 carbon atoms, or        straight-chain or branched alkynyl having 2 to 21000 carbon        atoms, and    -   R₂ is connected via a secondary or tertiary carbon atom to the        phenylene radical in the position ortho, meta or para to the SO₂        radical when Z is present, or to the SO₂ radical when Z is        absent, and is straight-chain or branched alkyl having 3 to 100        carbon atoms, straight-chain or branched alkenyl having 3 to 100        carbon atoms, straight-chain or branched alkynyl having 3 to 100        carbon atoms, straight-chain or branched alkoxy having 3 to 100        carbon atoms, straight-chain or branched hydroxyalkyl having 3        to 100 carbon atoms,        -   where the hydroxyl radical is connected via a primary or            secondary carbon atom to the alkyl radical, or        -   polyoxaalkyl having 3 to 100 carbon atoms.

Preferred compounds among these are, inter alia:

-   -   in which n and m are natural numbers and 3<(n+m)<60,        CH₂═CH—COO—SO₂—CH₂—CH═CH—C₁₃H₂₇,        CH₂═CH—COO—SO₂—O—C₁₂H₂₅,

andCH₂═CH—COO—SO₂—O—CH₂—CH₂—O—CH₂—CH₂—O—C₁₂H₂₅.Preparation and Investigation of the Compound

460 ml of distilled water are put into a 1000 ml Erlenmeyer flask andthen 94 g of sodium dodecylbenzenesulfonate are added. The mixture isstirred with a magnetic stirrer at about 50° C. until dissolution iscomplete, and then the solution is left to stand for about 15 minutes tocool. Then 2.4 ml of anhydrous acrylic acid are slowly added whilestirring, and the solution is stirred further until it reaches roomtemperature. After this solution has been left to stand in the dark atroom temperature for at least 24 hours, 79.5 ml of distilled water areput into a 250 ml Erlenmeyer flask, and then 0.5 ml of a 0.066 Mphosphate buffer solution (pH 7.00) is added while stirring.Subsequently, 1 g of the solution mixture obtained the previous day isadded while stirring. The resulting solution of the title compound has apH of about 4.6.

The following reagents are provided:

-   -   catalase from ox liver (as active endogenous catalase)    -   catalase from Aspergillus niger (as active intracellular        catalase)    -   catalase from microorganisms^((*)) (as active intracellular        catalase)    -   antifoam (RD® from Dow Corning)    -   hydrogen peroxide (Perhydrol® 30% from Merck) ^((*))various        bacteria and/or fungi

Each sample contains in each case 35 units of catalase from ox liver orfrom Aspergillus niger or from microorganisms per ml of distilled water.The 1 ml sample is put in a 15 ml tube (Vacutainer® 16×125 mm fromBecton Dickinson), and in each case 7 ml of the aforementioned solutionof the title compound are added and mixed therewith (Vortex® Genie 2mixer from Merck). Then 1 drop of said antifoam is added and mixing isrepeated. Thereafter 0.23 g of said hydrogen peroxide is added, and thetube is immediately made gas-tight with a stopper made ofchemical-resistant elastic plastic (Viton® from Du Pont de Nemours),briefly (for a few seconds) ventilated with the aid of a hollow needle(Ø1 mm hypodermic needle) piercing the stopper and, immediatelythereafter, closed gas-tight again by withdrawing the hollow needle fromthe stopper. The tube which has been closed gas-tight is left to standat room temperature for 15 minutes. The tube which has been tilted tothe horizontal is then shaken back and forth 10 times in order to ensurethat the cell membranes burst open. The pressure in the tube is thenmeasured (cf. the description hereinafter of the preferred pressuregage).

It is found that the pressure in the tube does not increase. It followsfrom this that the title compound inactivates both active endogenouscatalase and active intracellular catalase. It also follows from thisthat the title compound can be used to kill microorganisms, becausethese microorganisms whose intracellular catalase is inactivated diebecause of the absence of the action of this catalase.

Preparation and Investigation of the CompoundCH₂═CH—COO—SO₂—O—C₁₂H₂₅

200 ml of distilled water are put into a 500 ml Erlenmeyer flask, andthen 25 g of sodium lauryl sulfate are added. The mixture is stirredwith a magnetic stirrer at about 70° C. until dissolution is complete.Then, while maintaining the temperature, 1.07 g of polyacrylic acid(M_(r)=500000 to 1000000) are slowly added while stirring, and thesolution is stirred for a further 45 minutes. Subsequently, 5 ml of a0.25 M solution of disodium hydrogen phosphate dihydrate in distilledwater are added, and then 2 ml portions of the solution are metered inevery 15 minutes until a total of 12 ml of the solution has been used.The resulting solution mixture is initially stirred further whilemaintaining the temperature for 60 minutes and then cooled to roomtemperature. The solution mixture obtained in this way has a pH of about5.9. Then 42 ml of distilled water are put in a 250 ml Erlenmeyer flask,and thereafter 5 ml of a 0.066 M phosphate buffer solution (pH 7.00) areadded while stirring. Subsequently, 3 g of the solution mixture obtainedpreviously are added while stirring. The resulting solution of the titlecompound has a pH of about 6.4

The same reagents as previously described in the preceding text areprovided, and the catalase is exposed to the action of the titlecompound, and the catalase activity resulting thereafter is investigatedby means of the oxygen evolution (increasing pressure), in the same wayas in the preceding text.

If only active endogenous catalase is present in the sample, it is foundthat the pressure in the tube does not increase. If, however, activeintracellular catalase from microorganisms such as bacteria and/orfungi, especially molds and/or yeasts, is also present in the sample, itis found that the pressure in the tube increases. It follows from thisthat the title compound inactivates active endogenous catalase but doesnot noticeably inactivate active intracellular catalase.

Uses of the Compounds of the Invention

The preceding examples of the preparation and investigation of chemicalcompounds of the invention show that the latter bring about amodification of the kinetics of the enzymatic action of catalase. It hasemerged that a reversible or irreversible inactivation of the enzymaticaction of catalase results depending on the compound of the inventionused and depending on the microorganisms treated therewith, it beingpossible by suitable choice of the chemical compounds of the inventionto inactivate the enzymatic action either only of active endogenouscatalase or both of the latter and of active intracellular catalase, inparticular from live microorganisms such as bacteria and/or fungi, inparticular molds and/or yeasts.

Chemical compounds of the invention which irreversibly inactive theenzymatic action of active intracellular catalase of live microorganismssuch as bacteria and/or fungi, in particular molds and/or yeasts, bringabout the death of these microorganisms.

The procedure for using the chemical compounds of the invention formeasuring a concentration of live and/or active microorganisms such asbacteria and/or fungi, in particular molds or yeasts, is describedbelow.

Method for Measuring a Concentration of Live and/or ActiveMicroorganisms in a Sample Liquid

The method according to the invention for measuring a concentration oflive and/or active microorganisms in a sample liquid is described belowby means of FIGS. 1 to 8. It is based on the determination of theevolution of oxygen from hydrogen peroxide which is added to the sampleliquid, which has been diluted and/or buffered where appropriate, and isbroken down rapidly to oxygen and water by catalase present in thesample liquid.

FIG. 1 shows a provided essentially cylindrical container in oneembodiment as test tube 1 which is, for example, a 15 ml Vacutainer®16×125 mm tube from Becton Dickinson and can be closed gas-tight with astopper 2 which is made of chemical-resistant elastic plastic, forexample of Viton® from Du Pont de Nemours, and can be placed thereon.For illustration, the stopper 2 is depicted placed on the test tube 1 inFIG. 1.

FIG. 2 illustrates introduction of 1 ml of the sample liquid 3 to beinvestigated into the test tube 1. It is not depicted here that thesample liquid 3 has been obtained where appropriate from an originalsample by dilution and/or buffering (preferably to a pH between 6 and7).

FIG. 3 illustrates admixture of 7 ml of a reagent liquid 4 which is anapproximately 0.05 M aqueous solution of the active substance of theinvention, i.e. of a chemical compound of the invention or of a mixtureof chemical compounds of the invention, to the sample liquid 3. Theactive substance of the invention is chosen in this case, with a view tothe desired measurement of a concentration of live and/or activemicroorganisms, so that it inactivates the enzymatic action of anyendogenous catalase present in the sample liquid, but does notnoticeably inactivate the enzymatic action of intracellular catalase ofthe microorganisms to be measured.

FIG. 4 illustrates admixture, preferably by metered dropwise addition,of about 0.2 ml (about 0.23 g) of a 30% by weight aqueous hydrogenperoxide solution 5 to the amounts of liquids 3 and 4 illustrated inFIG. 3, after which a concentration of about 1% by volume of hydrogenperoxide is set up in the mixture 6 (illustrated in FIG. 5) resultingafter mixing has taken place. It is helpful where appropriate to add oneor a few drops of antifoam before addition of the hydrogen peroxidesolution.

FIG. 5 illustrates the stopper 2 being placed on the test tube 1immediately after the addition, illustrated in FIG. 4, of hydrogenperoxide solution 5, in order to close the mixture 6 gas-tight therein.

FIG. 6 illustrates piercing of the stopper 2, immediately after thegas-tight closure of the test tube 1 therewith illustrated in FIG. 5,with a hollow needle 7 which is, for example, a Ø1 mm hypodermic needle,after which the gas pressure prevailing in the test tube 1 equalsatmospheric pressure. After a few, preferably 1 to 2, seconds, thehollow needle 7 is withdrawn from the stopper 2, after which the lattercloses gas-tight, owing to its elasticity and because it is radiallycompressed in the region of the test tube 1, a (not depicted) piercedchannel left behind by the withdrawan hollow needle 7.

FIG. 7 illustrates the mixture 6 then remaining gas-tight in the closedtest tube 1 for a predetermined reaction time, which is counted from thewithdrawal of the hollow needle 7 from the stopper 2, preferably for 15minutes and, during this, being shaken in the test tube 1 which has beentilted horizontal, preferably by vigorous shaking back and forth 10times, which is symbolized by the double-headed arrow 8. During thisreaction time, oxygen is evolved from the hydrogen peroxide present inthe mixture 6.

FIG. 8 illustrates measurement of the pressure prevailing in the testtube 1 at the end of the predetermined reaction time by piercing thestopper 2 with a hollow needle 9, which is likewise, for example, a Ø1mm hypodermic needle, this hollow needle 9 being connected to adiagrammatically depicted pressure sensor 10. This pressure sensor 10 isin turn part of a pressure gage 11 which is described in detailhereinafter in connection with FIG. 9.

It is evident from comparison of FIGS. 6 and 8 that the stopper 2 ispierced by hollow needle 7 (for pressure equalization) and by hollowneedle 9 (for pressure measurement) at different places in order toavoid an escape of gas from test tube 1 when hollow needle 9 is insertedinto the (not depicted) pierced channel left behind in stopper 2 bywithdrawn hollow needle 7 (and measurement errors resulting therefrom).

When the method according to the invention is carried out, it isnormally known whether and in what ratio the investigated material orthe sample thereof has been diluted in order to afford the sample liquid3. It is also normally known, from experience, what types ofmicroorganisms prevail in the investigated material, so that theefficiency or the activity of the catalase occurring in the investigatedmaterial is normally also known. It is possible with the aid of thisinformation and after a blank and, with known concentrations ofmicroorganisms of the same type, a characteristic curve of the pressuregage 11 has been measured, to calculate the concentration of live and/oractive microorganisms in the sample from the measured pressure. It iseasy and requires no detailed description here to provide amicroprocessor 22 and data memory 23 in the pressure gage 11 (cf.concerning this the description hereinafter in connection with FIG. 9)in order to store said data therein and process it so that theconcentration of live and/or active microorganisms in the sample isautomatically calculated and preferably displayed directly as numericalvalue.

Apparatus for Measuring a Concentration of Live and/or ActiveMicroorganisms in a Sample Liquid

FIG. 9 illustrates a pressure gage, designated overall by 11, of anapparatus for carrying out the method of the invention. The pressuregage 11 includes a diagrammatically depicted pressure probe part,designated overall by 12, and a diagrammatically depicted pressureevaluation part, designated overall by 13, which are connected togetherby electric leads of a shielded multicore electric cable 14.

The pressure probe part 12 includes a base 15 in the form of acylindrical blind socket whose bore 16 is disposed with the axisvertically during use. The test tube 1 fits with little play in thisbore 16, and it can be introduced axially therein to about one half ofits length. An annular rim of the base 15 at the end of the bore 16forms a stop face 17 which is coordinated with the test tube 1, whichhas been completely inserted into the bore 16, and is in a predeterminedposition in relation to this tube, irrespective of whether and how far astopper 2 has been pushed into the test tube 1 in order to close thelatter. The pressure probe part 12 additionally includes the previouslymentioned hollow needle 9 and the previously mentioned pressure sensor10, and a cylindrical blind positioning socket 18 whose bore 19 isdisposed with the axis vertically during use. The hollow needles 9 arefirmly disposed, coaxially to the bore 19, on the pressure sensor 10,and the latter is firmly disposed on the positioning socket 18. Anannular rim of the positioning socket 18 at the end of the bore 19 formsa stop face 20 in fixed position and coordination with the positioningsocket 18 and consequently, via the pressure sensor 10 and the hollowneedle 9, with the tip 21 thereof. The axial length of the positioningsocket 18 is such that, on use of the pressure probe part 12, thestopper 2 is pierced by the hollow needle 9 to such an extent, and thelatter can be introduced into the interior of the test tube 1 only tosuch an extent, that its tip 21 remains above the mixture 6 present inthe test tube 1, in a headspace located there. Finally, as is evidentfrom FIG. 9, the pressure probe part 12, the test tube 1 and the stopfaces 17 and 20 coordinated therewith have dimensions and are positionedrelative to one another in such a way that, when the stopper 2 ispierced by the hollow needle 9, only the latter comes into contact withthe stopper 2, so that the latter is stressed only slightly and mainlyonly radially, and consequently is not pushed into the interior of thetest tube 1, which would cause a volume change and measurement errorsresulting therefrom, which are thus avoided.

The pressure evaluation part 13 includes a microprocessor 22 and datamemory 23, which receive data from the pressure sensor 10 in thepressure probe part 12 via cable 14. In addition, at least some of thedata memories 23 (for example as EPROM, flash memory or otherexchangeable and employable memories), and thus also the microprocessor22, are programmable. It is thus possible to store in the pressureevaluation part 13 data on the blank of the pressure gage 11 and data ona characteristic curve of the pressure gage 11. This characteristiccurve is determined with known concentrations of live and/or activemicroorganisms of the same type as those expected to be present in thesample liquid 3, and data on the sample dilution can also be subsumed inthe data on the characteristic curve of the pressure gage 11. Themicroprocessor 22 programmed in this way calculates a value of theconcentration of live and/or active microorganisms in the sample fromthe data on the pressure provided by the pressure probe part 12 via thecable 14, and from the data which the microprocessor 22 receives fromthe data memories 23. Owing to this automated and programmed calculationit is possible to obtain a calculated value which represents directly asnumerical value the concentration of live and/or active microorganismsin the sample and appears on a display 24 in the pressure evaluationpart 13.

FIG. 10 illustrates a diagrammatically depicted ventilation implement,designated overall by 32, of an apparatus for carrying out the methodaccording to the invention. Some parts of this ventilation implement 32are identical to corresponding parts in the pressure probe part 12, andthey may even be the same parts in a different function: such equivalentparts are only mentioned here, reference being made to the descriptiongiven hereinbefore of their equivalents in connection with the pressureprobe part 12.

The ventilation implement 32 includes a base 25 which is identical tothe base 15. The test tube 1 fits in the same way into a bore 26, whichis identical to the bore 16 and is likewise disposed with the axisvertical during use. An annular rim of the base 25 at the end of thebore 26 forms a stop face 27 which is identical to the stop face 17. Theventilation implement 32 additionally includes the previously mentionedhollow needle 7 and a cylindrical blind positioning socket 28, whosebore 29 is disposed with the axis vertical during use. The hollow needle7 is firmly disposed on the positioning socket 28 parallel to the axisof the bore 29 but not coaxial thereto, i.e. eccentrically. An annularrim of the positioning socket 28 at the end of the bore 29 forms a stopface 30 in fixed position and coordination with the positioning socket28 and consequently, via the hollow needle 7, with its tip 31. The axiallength of the positioning socket 28 is such that, on use of theventilation implement 32, the stopper 2 is pierced by the hollow needle7 only to the extent, and the latter can be introduced into the interiorof the test tube 1, only to the extent, that its tip 31 remains abovethe mixture 6 present in the test tube 1 in a headspace present therein.Finally, as is evident from FIG. 10, the ventilation implement 32, thetest tube 1 and the stop faces 27 and 30 coordinated therewith havedimensions and are positioned relative to one another such that, whenthe stopper 2 is pierced by the hollow needle 7, only the latter comesinto contact with the stopper 2, so that the latter is stressed onlyslightly and mainly only radially, and consequently is not pushed intothe interior of the test tube 1, which would cause a volume change andmeasurement errors resulting therefrom, which are thus avoided.

FIG. 11 illustrates details of the stopper 2 depicted diagrammaticallyin axial longitudinal section. In order to ensure that the stopper 2 onthe one hand is able to close the test tube 1 gas-tight, but on theother hand can be pierced by the hollow needles 7 and 9, this stopper 2is produced from a chemical-resistant elastic plastic and is designed tobe substantially mushroom-shaped, having a cylinder 33 which can beintroduced into the test tube 1 and on which are shaped a head 34 at thetop and a socket-like sealing lip 35 at the bottom. When the test tube 1is closed with the stopper 2, the cylinder 34 and the sealing lip 35 arelocated, radially compressed, completely inside the test tube 1, whereasthe somewhat wider head 33 remains outside the test tube 1 and coversits mouth. If, for example, the test tube 1 is a 15 ml Vacutainer®16×125 mm tube from Becton Dickinson, and the stopper 2 consists ofViton® from Du Pont de Nemours, the total height of the stopper 2 to bepierced by the hollow needles 7 and 9 is about 5 mm, and the cylinder 34is radially compressed by about 7%.

The invention has been described above by means of individual examplesrelating to the preparation of the substances of the invention and thebringing about and/or use of a modification of the kinetics of theenzymatic action of catalase. There are many possible uses of thesubstances of the invention, of the use thereof for modifying thekinetics of the enzymatic action of catalase and, where appropriate, forkilling microorganisms, of the method according to the invention formeasuring a concentration of live and/or active microorganisms, and ofthe corresponding apparatus according to the invention. Mention may bemade, in a list of examples without any restriction thereto, as possibleuses for measurement, for monitoring and/or for process control, and forkilling microorganisms of: in the case of foodstuffs at the level ofconsumption, marketing and industry (against microbial spoilage); in thecase of processing of materials by cutting (in relation towater-miscible cooling lubricants etc.); in the case of human medicineand veterinary medicine, in particular in supply and disposal inhospitals and medical laboratories (blood, urine, facilities andinstruments, dressings etc.); in the supply of fresh water, in waterstorage and in sewage treatment; in ventilation and air-conditioningsystems (fresh air purification, air circulation, waste air cleanup);and in research and development in the aforementioned or other areas.

In summary, it can be stated that with the catalase method according tothe invention which has been depicted and described

-   -   a) even very low microbe counts can be detected from a sample by        filtration using suitable membrane filters;    -   b) a differentiation is possible between bacteria on the one        hand, fungi and yeasts on the other hand, by filtration using        suitable membrane filters; and    -   c) a greater sensitivity of measurements is achieved because        more sensitive pressure transducer probes than previously can be        used;        and that the compound of the invention inactivates both active        endogenous catalase and active bacterial catale, and can be used        as active disinfection component alone or for further        incorporation in disinfectants for killing microorganisms.

LIST OF REFERENCE NUMBERS

-   1 test tube-   2 stopper-   3 sample liquid-   4 reagent liquid-   5 hydrogen peroxide solution-   6 mixture-   7 hollow needle for pressure equalization-   8 shaking back and forth-   9 hollow needle for pressure measurement-   10 pressure sensor-   11 pressure gage-   12 pressure probe part-   13 pressure evaluation part-   14 electric cable-   15 base of the pressure probe part 12-   16 bore in the base 15-   17 stop face on the base 15-   18 positioning socket-   19 bore of the positioning socket 18-   20 stop face on the positioning socket 18-   21 tip of the hollow needle 9-   22 microprocessor-   23 data memory-   24 display-   25 base of the ventilation implement 32-   26 bore in the base 25-   27 stop face on the base 25-   28 positioning socket-   29 bore of the positioning socket 28-   30 stop face on the positioning socket 18-   31 tip of the hollow needle-   32 ventilation implement-   33 cylinder of the stopper 2-   34 head of the stopper 2-   35 sealing lip of the stopper 2

1. A sulfonyl ester selected from the group consisting of:

in which n and m are natural numbers and 3<(n+m)<60,

and CH₂═CH—COO—SO₂—O—CH₂—CH₂—O—CH₂—CH₂—O—C₁₂H₂₆.
 2. A method ofmodifying the kinetics of the enzymatic action of catalase by mixingsaid catalase with a compound as claimed in claim
 1. 3. The method asclaimed in claim 2, characterized in that the modification of thekinetics is an inactivation.
 4. The method as claimed in claim 3,characterized in that the inactivation is irreversible.
 5. The method asclaimed in claim 4, characterized in that the inactivation relates tothe enzymatic action of active endogenous catalase.
 6. The method asclaimed in claim 4, characterized in that the inactivation relates tothe enzymatic action of active endogenous catalases.
 7. The method asclaimed in claim 6, characterized in that the inactivation relates tothe enzymatic action of active endogenous catal ass without a noticeableinactivation of the enzymatic action of active intracellular catalasetaking place.
 8. The method as claimed in claim 4, characterized in thatthe inactivation relates to the enzymatic action of active endogenouscatalase without a noticeable inactivation of the enzymatic action ofactive intracellular catalase taking place.
 9. The method as claimed inclaim 8, characterized in that the active intracellular catalase is thatof live microorganisms.
 10. The method as claimed in claim 7,characterized in that the active intracellular catalese is that of livemicroorganisms.
 11. The method as claimed in claim 10, wherein the livemicroorganisms is selected from the group consisting of bacteria andfungi, Including molds and yeasts.
 12. The method as claimed in claim 9,wherein the live microorganisms is selected from the group consisting ofbacteria and fungi, including molds and yeasts.
 13. The method asclaimed in claim 12 for killing microorganisms.
 14. The method asclaimed in claim 11 for killing microorganisms.
 15. The method asclaimed in claim 10 for killing microorganisms.
 16. The method asclaimed in claim 9 for killing microorganisms.
 17. The method as claimedin claim 8 for killing microorganisms.
 18. The method as claimed inclaim 7 for killing microorganisms.
 19. The method as claimed in claim 6for killing microorganisms.
 20. The method as claimed in claim 5 forkilling microorganisms.
 21. The method as claimed in claim 4 for killingmicroorganisms.
 22. The method as claimed in claim 3 for killingmicroorganisms.
 23. The method as claimed in claim 2 for killingmicroorganisms.